Ube  ittntverattB  of  Chicago 


MO  * 


I 


FERTILIZATION  IN  LILIUM 


A DISSERTATION 

SUBMITTED  TO  THE  FACULTY 
OF  THE  OGDEN  GRADUATE  SCHOOL  OF  SCIENCE 
IN  CANDIDACY  FOR  THE  DEGREE  OF  -V 
DOCTOR  OF  PHILOSOPHY 

DEPARTMENT  OF  BOTANY 


BY 

WANDA  WENIGER 


Private  Edition,  Distributed  By 
THE  UNIVERSITY  OF  CHICAGO  LIBRARIES 
CHICAGO,  ILLINOIS 


Reprinted  from 

The  Botanical  Gazette,  Vol.  LXVI,  No.  3 
September  1918 


Cbe  ^University  of  Chicago 


FERTILIZATION  IN  LILIUM 


A DISSERTATION 

SUBMITTED  TO  THE  FACULTY 
OF  THE  OGDEN  GRADUATE  SCHOOL  OF  SCIENCE 
IN  CANDIDACY  FOR  THE  DEGREE  OF 
DOCTOR  OF  PHILOSOPHY 

DEPARTMENT  OF  BOTANY 


BY 

WANDA  WENIGER 


Private  Edition,  Distributed  By 
THE  UNIVERSITY  OF  CHICAGO  LIBRARIES 
CHICAGO,  ILLINOIS 

Reprinted  from 

The  Botanical  Gazette,  Vol.  LXVI,  No.  3 
September  1918 


Digitized  by  the  Internet  Archive 
in  2017  with  funding  from 

University  of  Illinois  Urbana-Champaign  Alternates 


https://archive.org/details/fertilizationinlOOweni 


ST/,  3 


a, 

FERTILIZATION  IN  LILIUM 


% 


10 

fv 

* 


^ CONTRIBUTIONS  FROM  THE  HULL  BOTANICAL  LABORATORY  243 

Wanda  Weniger 

s*  . 

^ (with  plates  xi-xiii) 

Introduction 

The  cytological  phenomena  of  fertilization  have  been  studied 
with  greater  detail  in  the  gymnosperms  than  in  the  angiosperms. 
This  paper  is  the  result  of  an  attempt  to  discover  whether  there 
is  a similarity  between  the  process  of  fertilization  as  already 
described  for  gymnosperms  and  that  of  angiosperms.  Lilium  has 
long  been  the  type  used  in  the  study  of  fertilization  in  the  class- 
room; it  was  chosen  for  the  subject  of  study  in  this  case  because  it 
lends  itself  particularly  well  to  cytological  work.  The  writer  is 
indebted  to  Dr.  C.  J.  Chamberlain  for  the  suggestion  of  the 
problem  and  for  his  helpful  assistance  throughout  the  progress 
of  the  work. 

In  Pinus  (1,  3,  5,  7,  8),  Tsuga  (14),  Juniperus  (18,  19),  and 
Abies  (12)  evidence  has  been  brought  to  bear  upon  the  fact  that  no 
fusion  of  the  male  and  female  chromatic  substance  takes  place. 

Blackman  (i),  in  1898,  described  the  cytological  features  of 
fertilization  in  Pinus  silvestris.  While  the  outlines  of  the  2 sexual 
nuclei  are  still  visible,  the  chromosomes  are  found  in  2 separate 
clumps;  and  even  on  the  spindle  fibers  of  the  first  division  they 
can  be  distinguished  into  2 groups.  After  a longitudinal  splitting 
the  half  chromosomes  fuse  together  in  the  telophase  of  the  division. 

Chamberlain’s  (3)  account  of  oogenesis  in  Pinus  Laricio 
includes  figures  of  the  male  and  egg  nuclei.  He  states  that  after 
the  male  pronucleus  is  within  the  oosphere  nucleus  the  chromatin 
of  the  2 pronuclei  appears  as  2 distinct  masses  in  the  spireme  stage. 
“ Perhaps  segmentation  of  the  2 spiremes  occurs  while  they  are  still 
separate.”  In  Tsuga  canadensis  Murrill  (14)  reports  2 sets  of 
chromosomes  distinct  in  the  equatorial  region  of  the  first  spindle. 
259l  [Botanical  Gazette,  vol.  66 


BOTANICAL  GAZETTE 


[SEPTEMBER 


260 


Miss  Ferguson  (7),  in  her  first  paper  on  Pinus  Strobus  in  1901, 
finds  that  the  2 chromatic  groups  are  distinctly  recognizable  at  the 
time  of  the  segmentation  of  the  spiremes,  and  can  still  be  clearly 
made  out  during  the  early  development  of  the  chromosomes,  but 
not  as  late  as  the  equatorial  plate  stage.  “ There  is  never  any 
fusion,  as  ordinarily  understood,  of  the  male  and  female  nuclei.” 
In  her  second  paper  on  Pinus  Miss  Ferguson  (8)  describes  the 
longitudinal  splitting  of  the  24  chromosomes  on  the  equatorial 
plate.  According  to  Noren  (18,  19),  the  essential  features  of  fer- 
tilization in  Juniperus  communis  are  similar  to  those  of  Pinus. 

A very  detailed  account  of  fertilization  is  given  by  Hutchinson 
(12)  for  Abies  balsamea.  Two  groups  of  chromatin  at  the  micro- 
pylar  end  of  the  egg  nucleus,  one  male  and  the  other  female,  become 
separated  into  16  chromosomes  each,  and  these  pass  on  to  the 
spindle  fibers.  The  2 spindles  in  the  metaphase  fuse,  and  the 
chromosomes  are  arranged  to  form  16  pairs,  each  pair  forming  a C, 
in  which  the  2 chromosomes  are  twisted  about  each  other.  By 
means  of  a transverse  break  at  the  angle  of  the  bent  chromosomes 
each  pair  forms  4 segments.  Of  the  64  segments,  32  go  to  each  pole, 
where  in  the  daughter  nuclei  they  remain  very  distinct. 

Chamberlain  counted  12  chromosomes  in  Stangeria  (4)  at 
the  equatorial  plate  stage  of  the  division  of  the  fertilized  egg,  while 
the  sporophyte  number  is  24.  He  accounts  for  the  haploid  number 
by  assuming  the  chromosomes  to  be  of  a double  character,  and 
supports  Hutchinson’s  view  of  the  pairing  of  chromosomes. 

In  angiosperms  the  behavior  of  the  chromatin  during  fertiliza- 
tion has  received  little  attention.  In  the  majority  of  cases  the 
statement  is  made  that  the  nuclei  fuse  while  in  the  resting  condition 
almost  immediately  after  they  come  in  contact  and  form  a definite 
resting  nucleus,  differing  only  in  its  greater  size  from  the  unfertilized 
egg  nucleus. 

Guignard’s  (9)  paper  in  1891  on  fertilization  in  Lilium  Martagon 
contains  statements  overlooked  by  most  writers.  The  formation 
of  2 distinct  spiremes  in  the  male  and  egg  nuclei  was  observed  but 
not  figured.  No  fusion  is  brought  about  between  the  chromatin 
of  the  2 nuclei,  even  when  the  nuclear  membranes  disappear. 
The  segments  of  each  spireme  pass  on  to  the  equatorial  plate,  where 


WEN IGER — TER 77 IJZA  TION 


1918] 


2 61 


each  splits  longitudinally.  In  1895  Mottier  (13)  first  described 
the  vermiform  shape  of  the  male  nuclei  in  Lilium  Martagon. 
The  male  and  egg  nuclei  fuse  in  the  resting  condition  after  com- 
ing in  contact  and  are  figured  as  forming  a resting  nucleus.  In 
1898  Nawaschin  (15)  announced  the  discovery  of  double  fertili- 
zation in  Lilium  Martagon  and  Fritillaria  tenella.  The  male 
nucleus  that  fuses  with  the  polar  nuclei  loses  its  spiral  form,  but 
the  3 nuclei  remain  distinct  until  the  prophase  of  the  division.  The 
fusion  of  the  3 nuclei  occurs  when  the  numerous  chromosomes  come 
together  on  the  equatorial  plate.  “ Fusion  occurs,  not  in  the  resting 
stage,  as  Mottier  indicates,  but  in  the  prophases  of  the  division, 
as  Guignard  first  observed.” 

The  motility  of  the  male  nuclei  is  described  for  Lilium  Martagon 
and  Fritillaria  tenella  by  Nawaschin  (16,  17);  for  the  tulip  by 
Guignard  (ii);  and  for  Lilium  Martagon  and  L.  auratum  by 
Blackman  and  Welsford  (2),  and  Miss  Welsford  (23).  These 
authors  attribute  independent  motion  to  the  male  nuclei. 

In  Paris  quadrifolia  and  Trillium  grandiflorum  Ernst  (6)  finds 
a striking  difference  between  the  fusion  of  the  male  nucleus  with  the 
egg  and  that  with  the  polar  nuclei.  In  the  former  case  the  fusion 
is  complete,  so  that  a typical  resting  nucleus  is  formed.  In  the 
latter  case  the  polar  nuclei  begin  to  form  spiremes  even  before  the 
male  nucleus  arrives,  and  in  the  group  of  the  3 nuclei  (the  2 polar 
nuclei  and  the  male  nucleus)  3 spiremes  are  distinguishable. 

Distinct  maternal  and  paternal  chromosomes  were  first  described 
for  an  angiosperm  by  Miss  Pace  (21).  She  found  spiremes  in  all 
nuclei  of  the  embryo  sac  of  Cypripedium  before  fusion  took  place. 
The  spireme  was  well  formed  in  every  nucleus,  and  shortened  almost 
enough  to  segment  into  chromosomes.  “It  would  seem  in  this 
case,  that  if  fusion  does  take  place,  there  could  be  no  possibility 
of  a fusion  of  the  chromatin,  which  would  certainly  divide  into 
chromosomes  from  the  spireme  as  it  is  now  formed.” 

Nawaschin  (17)  published  another  paper  on  Lilium  Martagon 
in  1910,  again  emphasizing  the  fact  that  the  mature  nuclei  are 
capable  of  movement.  He  finds  that  the  mitosis  of  the  2 male 
nuclei  in  the  pollen  tube  is  characterized  at  an  early  stage  by  sharply 
differentiated  chromosomes,  so  that  the  sperm  nuclei  do  not  reach 


262 


BOTANICAL  GAZETTE 


[SEPTEMBER 


the  resting  stage,  but  remain  in  the  condition  characteristic  of  a 
telophase. 

Recently  Sax  (22)  has  investigated  Fritillaria  pudica.  In  most 
cases  it  is  not  until  the  male  nucleus  and  the  egg  nucleus  have 
completely  fused  that  he  finds  any  appearance  of  the  formation  of 
the  spireme.  In  rare  cases,  however,  the  spireme  stage  is  found 
while  the  2 nuclei  are  still  distinct  in  outline.  He  believes  that  the 
rare  appearance  of  such  cases  is  probably  of  little  significance,  since 
it  is  probable  that  these  nuclei  subsequently  fuse  completely  because 
no  later  stages  were  found  in  which  fusion  was  incomplete.  From 
the  many  stages  and  abundant  cases  of  triple  fusion  he  observed 
he  thinks  there  is  no  doubt  that  the  2 polar  nuclei  and  the  male 
nucleus  fuse  completely  and  that  the  subsequent  division  is  normal. 

Methods 

Stages  in  fertilization  were  obtained  from  ovaries  of  Lilium 
philadelphicum  collected  in  the  field  near  Osborn,  Calumet,  and 
Pine,  Indiana,  in  June  and  early  July,  1916,  at  the  time  when  the 
petals  “ snapped,”  and  after  the  petals  had  fallen.  To  correlate 
the  time  of  pollination  with  stages  in  fertilization,  flowers  were 
brought  into  the  laboratory,  pollinated,  and  kept  under  bell  jars 
for  several  days,  until  fertilization  had  taken  place.  In  general, 
it  may  be  said  that  the  petals  drop  on  the  third  day  after  pollination, 
and  the  style  separates  from  the  ovary  on  the  fourth  or  fifth  day. 
The  male  nucleus  was  in  contact  with  the  egg  nucleus  from  60  to  72 
hours  after  pollination. 

The  material  for  Lilium  longiflorum  was  obtained  from  plants 
grown  in  the  greenhouse.  It  produced  seeds  readily,  although  it  is 
generally  reported  not  to  set  seed.  The  male  nucleus  was  in  contact 
with  the  egg  nucleus  about  120  hours  after  pollination.  Of  the 
upward  of  500  cases  of  fertilization  observed  in  these  2 species,  the 
majority  showed  the  male  and  egg  nuclei  in  contact,  with  their 
chromatin  in  early  prophases  of  the  division. 

Chrom-acetic-osmic  and  Flemming’s  medium  solutions  were 
used  as  fixatives,  and  the  ovaries  trimmed  so  as  to  permit  more 
rapid  penetration  of  the  embryo  sacs.  Sections  were  cut  10  /x 
thick  and  stained  with  Flemming’s  triple  stain  or  Haidenhain’s 
iron-alum-haematoxylin. 


W ENIGER — FER  T I LIZ  A TION 


263 


1918] 


Observations 

Upon  leaving  the  pollen  tube  the  male  nuclei  retain  their  coiled 
shape  for  some  time.  The  egg  nucleus  (fig.  1),  with  chromatin  in  a 
resting  condition  before  the  arrival  of  the  male  nucleus,  remains 
in  this  condition,  while  the  male  nucleus  lies  in  contact  with  it. 
Stages  can  be  found  abundantly  in  which  the  male  nucleus  has 
penetrated  the  egg  and  lies  adjacent  to  the  egg  nucleus,  and  in 
which  the  chromatin  of  the  former  is  in  an  early  prophase  (fig.  2), 
or  spireme  stage  (fig.  3),  while  the  chromatin  of  the  latter  more 
lightly  staining  nucleus  is  in  the  resting  stage.  The  male  nucleus 
is  mor'e  or  less  curved  around  one  side  of  the  egg  nucleus  and  usually 
measures  about  9 ju  at  its  short  diameter,  while  the  spherical  egg 
nucleus  is  10-12  /x  in. diameter.  Soon  the  male  nucleus  becomes 
more  rounded,  as  is  shown  in  fig  4,  where  the  chromatin  in  both 
nuclei  is  still  in  the  same  stage  as  in  fig.  3. 

The  chromatin  of  the  egg  nucleus  is  then  formed  into  a spireme 
(figs.  5,  6);  but  this  spireme  was  never  found  to  stain  as  densely 
or  become  as  regular  as  that  of  the  male  nucleus.  The  membranes 
of  the  2 nuclei  seem  still  to  be  in  contact  at  this  stage.  No  fusion 
of  the  spiremes  takes  place,  but  each  is  segmented  into  chromo- 
somes independently.  This  account  agrees  with  that  of  Guignard 
for  Lilium  Martagon,  where  no  fusion  takes  place  between  the 
chromatic  elements  of  the  2 nuclei. 

In  the  gymnosperms  investigated  the  separate  groups  of  chro- 
mosomes formed  from  the  male  and  female  spiremes  respectively 
become  oriented  on  separate  spindles,  and  then  the  2 spindles  fuse 
during  the  metaphase.  Whether  or  not  this  is  true  for  Lilium 
has  not  been  determined.  The  entire  process  of  fertilization  in 
Lilium  is  an  exceedingly  rapid  one,  since  the  time  elapsing  between 
the  discharge  of  the  male  nuclei  and  the  formation  of  the  2-celled 
embryo  is  probably  not  longer  than  8 hours.  Since  the  contact 
stage  of  the  2 nuclei  in  the  prophases  of  the  division  is  of  such  rela- 
tively common  occurrence  in  preparations  made,  it  would  seem  that 
it  occupies  the  greater  part  of  this  time,  and  that  for  this  reason  the 
actual  division  of  the  fertilized  egg  is  a very  difficult  stage  to  obtain. 
One  very  favorable  preparation  shows  this  division,  with  some  of 
the  chromosomes  still  on  the  equatorial  plate  and  others  already 


BOTA  NIC A L GA ZETTE 


[SEPTEMBER 


264 

near  the  poles  of  the  spindle.  Figs.  1 1,  12,  and  13  represent  the 
3 sections  of  this  spindle,  and  in  fig.  14  the  3 drawings  are 
superimposed  and  slightly  diagrammed.  Of  the  24  chromosomes 
present  on  the  equatorial  plate,  12  are  contributed  by  the  male 
nucleus  and  12  by  the  egg  nucleus.  The  chromosomes  are  not 
drawn  into  the  sharp  U’s  and  C’s  so  characteristic  of  divisions  in 
Lilium.  The  chromosomes  come  together  in  pairs  in  which  they 
twist  more  or  less  about  each  other  (fig.  12 a).  Each  of  the  chromo- 
somes of  the  12  pairs  then  breaks  transversely  at  the  center  of  the 
ellipse  it  forms,  each  pair  giving  rise  to  4 segments.  The  48  seg- 
ments in  the  form  of  small  rods  remain  paired  (fig.  12 b,  c ) as  they 
move  toward  the  poles  of  the  spindle.  The  components  of  each 
pair  are  similar  in  size  so  far  as  could  be  determined;  one  segment 
is  male  and  the  other  female  in  origin.  In  fig.  14  the  12  pairs  of 
chromosomes  are  represented,  with  the  4 segments  of  a pair  indi- 
cated by  the  same  number.  All  segments  going  to  one  pole  are  in 
black,  those  to  the  opposite  pole  in  outline.  Chromosomes  8 and 
12  have  not  as  yet  come  in  contact  and  the  transverse  break  has 
not  yet  appeared.  This  behavior  of  chromosomes  resembles  that 
of  the  first  reduction  division  in  tetrad  formation.  There  is  a 
pairing  of  chromosomes  and  a subsequent  transverse  breaking. 
The  result  of  the  division  is  not  the  reduced  number  of  chromosomes, 
however,  but  the  diploid  number,  for  only  a transverse  break  occurs, 
and  no  further  splitting. 

In  the  telophase  of  this  division  (figs.  15,  16)  no  further  evidence 
of  the  pairing  of  the  chromosomes  could  be  observed.  It  would 
seem  probable  that  the  individuality  of  the  chromosomes  derived 
from  the  male  and  egg  nuclei  would  persist.  The  second  division 
(fig.  17)  of  the  fertilized  egg  is  in  all  respects  like  the  ordinary  vege- 
tative division  in  Lilium , with  a longitudinal  splitting  of  the 
characteristic  U-shaped  chromosomes  during  the  metaphase. 

Observations  were  also  made  on  the  behavior  of  the  chromatin 
during  triple  fusion.  The  process  occurs  much  more  rapidly  and  the 
resulting  nucleus  divides  at  least  twice  before  the  fertilized  egg 
undergoes  division.  At  the  time  that  the  endosperm  nucleus 
divides  (fig.  7)  the  male  and  egg  nuclei  are  still  in  the  stage  shown  in 
fig.  3 or  4.  The  2 polar  nuclei,  with  membranes  distinct,  are  in  the 
resting  condition  when  the  male  nucleus  in  the  spireme  stage  comes 


WENIGER — FER  T1  LIZA  TION 


265 


1918] 


in  contact  with  them  (fig.  8).  A spireme  is  then  formed  in  each 

t polar  nucleus  also  (fig.  9),  and  the  nuclear  membranes  disappear 

at  the  point  of  contact  of  the  nuclei.  The  lower  polar  nucleus  is 
usually  a little  larger  than  the  one  coming  from  the  micropylar  end 

* of  the  embryo  sac.  The  male  nucleus  is  at  the  left  in  fig.  9. 

Segmentation  of  spiremes  occurs  so  that  on  the  spindle  (fig.  19) 
the  chromosomes  are  extremely  long  and  U-shaped.  The  number 
of  segments  is  difficult  to  ascertain,  but  it  approaches  the  3X 
number.  The  division  is  accomplished  by  a longitudinal  splitting 
of  chromosomes,  producing  in  the  anaphase  a mass  o"  long  bent 
segments  that  cannot  be  counted  with  any  satisfaction. 

There  is  a striking  difference  between  the  first  division  of  the 
fertilized  egg  and  that  of  the  endosperm  nucleus.  The  former  is 
characterized  by  shorter  straighter  chromosomes,  a pairing  of 
chromosomes,  and  a subsequent  transverse  breaking  of  ea:h  pair  to 
form  4 segments,  of  which  2 go  to  each  pole.  The  division  of  the 

* endosperm  nucleus  resembles  the  ordinary  vegetative  division  by 
means  of  a longitudinal  splitting  of  chromosomes.  The  number  of 
chromosomes  is  3#.  Since  previous  cytological  work  has  not 

► covered  the  necessary  phases,  it  is  possible  that  the  description  of 

the  behavior  of  chromosomes  during  the  first  division  of  the 
fertilized  egg  here  given  may  apply  quite  generally  to  angiosperms. 
A longitudinal  splitting  of  chromosomes  on  the  equatorial  plate 
would  bring  about  the  same  result  in  that  the  2X  number  of  chromo- 
somes goes  to  each  of  the  daughter  nuclei;  but  the  supposition  of  a 
longitudinal  splitting  would  not  account  for  the  situation  described. 
If  a longitudinal  splitting  should  occur  before  the  transverse  break- 
ing, rather  than  a pairing  of  chromosomes,  the  resulting  number 
would  be  96  rather  than  48  segments. 

The  3 phases  of  fertilization,  union  of  cells,  union  of  nuclei,  and 
union  of  chromosomes,  occur  in  rapid  succession  in  animals,  since 
the  reduction  division  immediately  precedes  fertilization.  In  plants 
the  3 processes  may  be  separated  for  a longer  or  shorter  period. 
* In  the  rusts  there  is  a long  gap  between  the  union  of  the  gametes 

at  the  base  of  the  aecidium  and  the  nuclear  and  chromosome 
conjugation.  In  some  of  the  green  algae,  such  as  Oedogonium,  the  3 
f come  close  together,  since  reduction  follows  soon  after  fertilization. 

In  the  brown  algae  the  3 are  also  close  together,  but  reduction 


266 


BOTANICAL  GAZETTE 


[SEPTEMBER 


precedes  fertilization.  In  the  higher  plants  cell  and  nuclear 
union  have  been  thought  to  come  close  together  at  the  beginning 
of  the  sporophyte  generation,  while  the  chromosome  union  did  not 
occur  until  during  the  reduction  division  at  the -end  of  the  sporo- 
phyte generation.  In  Abies , as  found  by  Hutchinson;  in 
Stangeria , according  to  Chamberlain;  and  in  Lilium  there  seems 
to  be  evidence  of  a chromosome  union  at  the  time  of  fertilization. 

Summary 

1.  The  egg  nucleus  is  in  a resting  condition  when  the  male 
nucleus,  in  spireme  stage,  comes  in  contact  with  it. 

2.  Distinct  male  and  female  spiremes  are  formed  which  are 
segmented  into  chromosomes  while  the  nuclei  are  in  contact. 

3.  On  the  equatorial  plate  the  male  and  female  chromosomes 
come  together  in  x number  of  pairs  and  divide  by  means  of  a 
transverse  break,  each  pair  forming  4 segments.  The  segments 
move  to  the  poles  in  pairs.  Of  the  4X  segments  formed,  2x  go  to 
each  pole  of  the  spindle. 

4.  The  chromosomes  on  the  equatorial  plate  of  the  second 
division  of  the  fertilized  egg  divide  longitudinally. 

5.  The  endosperm  nucleus  divides  at  least  twice  before  the 
fertilized  egg  undergoes  division. 

6.  A distinct  spireme  is  formed  in  each  of  the  nuclei  of  the  triple 
fusion,  and  the  $x  segments  are  oriented  on  the  equatorial  plate. 

7.  The  endosperm  nucleus  divides  in  the  typical  vegetative 
manner  by  means  of  a longitudinal  splitting  of  the  chromosomes. 

Experiment  Station 

Agricultural  College,  N.D. 


LITERATURE  CITED 

1.  Blackman,  V.  H.,  On  the  cytological  features  of  fertilization  and  related 
phenomena  in  Pinus  silvestris  L.  Phil.  Trans.  Roy.  Soc.  London  B 190: 
395-426.  pis.  12-14.  1898. 

2.  Blackman,  V.  H.,  and  Welsford,  E.  J.,  Fertilization  in  Lilium.  Ann. 
Botany  27: m-114.  pi.  12.  1913. 

3.  Chamberlain,  C.  J.,  Oogenesis  in  Pinus  Laricio.  Bot.  Gaz.  27:268-280. 
pis.  4-6.  1899. 

4.  , Stangeria  paradoxa.  Bot.  Gaz.  61:353-372.  pis.  24-26.  1916. 


5.  Dixon,  H.  N.,  Fertilization  of  Pinus  silvestris.  Ann.  Botany  8:21-34. 
pis.  3-5.  1894. 

6.  Ernst,  A.,  Chromosomenreduction,  Entwickelung  des  Embryosackes  und 
Befruchtung  bei  Paris  quadrifolia  L.  und  Trillium  grandiflorum  Salisb. 
Flora  91:1-46.  pis.  1-6.  1902. 

7.  Ferguson,  Margaret  C.,  The  development  of  the  egg  and  fertilization 
in  Pinus  Strobus.  Ann.  Botany  15:435-478.  pis.  23-25.  1901. 

8.  — , Contributions  to  the  life  history  of  Pinus , with  special  reference  to 

sporogenesis,  the  development  of  the  gametophytes,  and  fertilization. 
Proc.  Wash.  Acad.  Sci.  6:1-202.  pis.  1-24.  1904. 

9.  Guignard,  L.,  Nouvelles  etudes  sur  la  fecondation.  Ann.  Sci.  Nat.  Bot. 
VII.  14:163-296.  pis.  9-18.  1891. 

10.  , Sur  les  antherozoides  et  la  double  copulation  sexuelle  chez  les 

vegetaux  angiospermes.  Compt.  Rend.  128:864-871.  fig.  19.  1899;  Rev. 
Gen.  Bot.  11:129-135.  pi.  1.  1899. 

11.  , L’appareil  sexuel  et  la  double  fecondation  dans  les  Tulipes.  Ann. 

Sci.  Nat.  Bot.  VIII.  11:365-387 .pls.9-11.  1900. 

12.  Hutchinson,  A.  H.,  Fertilization  in  Abies  balsamca.  Bot.  Gaz.  60:457- 
472.  pis.  16-20.  1915. 

13.  Mottier,  D.  M.,  Uber  das  Verhalten  der  Kerne  bei  der  Entwickelung  des 
Embryosackes  und  die  Vorgange  bei  der  Befruchtung.  Jahrb.  Wiss.  Bot. 
31:125-158.  pis.  2,  3.  1898. 

14.  Murrill,  W.  A.,  The  development  of  the  archegonium  and  fertilization 
in  the  hemlock  spruce  ( Tsuga  canadensis).  Ann.  Botany  14:583-607. 
pis.  31,  32.  1900. 

15.  Nawaschin,  S.,  Resultate  einer  Revision  der  Befruchtungsvorgange  bei 
Lilium  Martagon  und  Fritillaria  tenella.  Bull.  Acad.  Imp.  Sci.  St.  Peters- 
bourg  9:377-382.  1898;  reviewed  in  Bot.  Centralbl.  78: 241-245.  1899. 

16.  , Neue  Beobachtungen  liber  Befruchtung  bei  Fritillaria  tenella  und 

Lilium  Martagon.  Bot.  Centralbl.  77:62.  1899. 

17.  , Naheres  liber  die  Bildung  der  Spermakerne  bei  Lilium  Martagon. 

Ann.  Jard.  Bot.  Buitenzorg.  II.  Supplement  III.  871-904.  pis.  33,  34.  1910. 

18.  Noren,  C.  0.,  Uber  Befruchtung  bei  Juniperus  communis.  Vorlaufige 
Mitteilungen.  Arkiv.  Bot.  Svensk.  Vetensk.  Akad.  3:pp.  n.  1904. 

19- , Zur  Entwickelungsgeschichte  des  Juniperus  communis.  Uppsala 

Universitets^Arsskrift  1907:  pp.  64.  pis.  4. 

20.  Overton,  E.,  Beitrag  zur  Kenntnis  der  Entwickelung  und  Vereinigung  der 
Geschlechtsproducte  bei  Lilium  Martagon.  Festschrift  (Nageli  und 
Kolliker).  Wtirzburg.  1891. 

21.  Pace,  Lula,  Fertilization  in  Cypripedium.  Bot.  Gaz.  44:353-374. 
pis.  24-27.  1907. 

22.  Sax,  Karl,  Fertilization  in  Fritillaria  pudica.  Bull.  Torr.  Bot.  Club 
43:505-522-  pis.  27-29.  1916. 

23.  Welsford,  E.  J.,  The  genesis  of  the  male  nuclei  in  Lilium.  Ann.  Botany 
28:265-2-70.  pis.  16,17.  1914. 


268 


BOTANICAL  GAZETTE 


[SEPTEMBER 


EXPLANATION  OF  PLATES  XI-XIII 

All  drawings  were  made  with  an  Abbe  camera  lucida  at  table  level  and 
Zeiss  apochromatic  objectives  and  compensating  oculars.  For  fig.  7 the  8 
ocular  and  2.0mm.  objective  were  used,  giving  a magnification  of  1500;  for 
the  remainder  of  the  drawings  the  18  ocular  was  used  with  the  2.0mm. 
objective,  and  the  magnification  was  4000.  All  drawings  were  reduced  one-half 
in  reproduction. 

PLATE  XI 

Fig.  1. — Egg  before  fertilization,  with  nucleus  in  resting  condition; 
X2000;  L.  philadelphicum. 

Fig.  2. — Male  nucleus  in  early  prophase ; egg  nucleus  in  resting  condition; 
X2000;  L.  philadelphicum. 

Fig.  3. — Chromatin  of  egg  nucleus  more  irregular;  male  nucleus  still 
curved  around  egg  nucleus;  X2000;  L.  philadelphicum. 

Fig.  4. — Male  nucleus  rounded;  X2000;  L.  longijlorum. 

Fig.  5. — Early  spireme  in  egg  nucleus ; X2000;  L.  philadelphicum. 

Fig.  6. — Distinct  spiremes  in  male  and  egg  nucleus;  segmentation  begun; 
X2000;  L.  longijlorum. 

Fig.  7. — Endosperm  nucleus  undergoing  division  while  egg  nucleus  is  in 
resting  stage  and  male  nucleus  in  contact  with  it  shows  a spireme;  X750; 
L.  philadelphicum. 

PLATE  XII 

Fig.  8. — Triple  fusion:  male  nucleus  in  spireme  stage,  upper  and  lower 
polar  nuclei  with  chromatin  in  resting  stage;  X2000;  L.  longijlorum. 

Fig.  9. — Distinct  spiremes  in  3 nuclei  of  triple  fusion;  male  nucleus  at 
upper  left;  X2000;  L.  philadelphicum. 

Fig.  10. — Metaphase  of  endosperm  nucleus;  X2000;  L.  philadelphicum. 

Fig.  17. — Second  division  of  fertilized  egg;  X2000;  L.  longijlorum. 

PLATE  XIII 

Figs,  i 1-13. — Three  sections  of  spindle  of  fertilized  egg  in  division,  showing 
pairing  of  chromosomes,  transverse  break,  moving  of  pairs  to  the  poles  ; X 2000; 
L.  longijlorum. 

Fig.  14. — Diagram  of  division  of  fertilized  egg,  made  by  superimposing 
figs.  11-13;  the  12  pairs  of  chromosomes  are  represented,  with  4 segments  of  a 
pair  indicated  by  the  same  number;  segments  in  solid  black  go  to  one  pole, 
while  those  in  outline  go  to  the  other  pole;  chromosomes  numbered  8 and  12 
have  not  yet  paired  or  segmented. 

Fig.  15. — Early  telophase  of  first  division;  X2000;  L.  longijlorum. 

Fig.  16. — Late  telophase  of  first  division;  X2000;  L.  longijlorum. 


BOTANICAL  GAZETTE,  LXVI 


PLATE  XI 


WENIGER  on  LILIUM 


4 


BOTANICAL  GAZETTE,  LXVI 


PLATE  XII 


WENIGER  on  LILIUM 


BOTANICAL  GAZETTE , LXVI 


Pi  ATE  XIII 


WENIGER  on  LILIUM 


L 


